Functional interrogation of the kinome using nucleotide acyl phosphates
Functional interrogation of the kinome using nucleotide acyl phosphates
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DOI：
10.1021/bi062142x
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发表时间：
2007-01-16
期刊：
影响因子：
2.9
通讯作者：
Kozarich, John W.
中科院分区：
文献类型：
--
作者：
Patricelli, Matthew P.;Szardenings, A. Katrin;Kozarich, John W.
蛋白激酶在信号转导途径中的核心作用引起了人们对针对这些酶进行广泛的治疗适应症的浓厚兴趣。在这里，我们报告了一种识别和定量任何物种的任何生物样本或组织中蛋白激酶的方法。该过程依赖于由生物素衍生物和 ATP 或 ADP 制备的含酰基磷酸的核苷酸。酰基磷酸探针选择性地与至少 75% 已知人类蛋白激酶的 ATP 结合位点发生共价反应。捕获来自标记蛋白质组的生物素化肽片段，然后使用基于质谱的分析平台进行测序和鉴定，以确定存在的激酶及其相对水平。此外，可以评估探针和抑制剂之间的直接竞争，以确定抑制剂对天然蛋白激酶以及数百种其他 ATP 酶的效力和选择性。广泛分析天然蛋白质组中激酶活性的能力为靶点发现和抑制剂选择性分析提供了令人兴奋的前景。
The central role of protein kinases in signal transduction pathways has generated intense interest in targeting these enzymes for a wide range of therapeutic indications. Here we report a method for identifying and quantifying protein kinases in any biological sample or tissue from any species. The procedure relies on acyl phosphate-containing nucleotides, prepared from a biotin derivative and ATP or ADP. The acyl phosphate probes react selectively and covalently at the ATP binding sites of at least 75% of the known human protein kinases. Biotinylated peptide fragments from labeled proteomes are captured and then sequenced and identified using a mass spectrometry-based analysis platform to determine the kinases present and their relative levels. Further, direct competition between the probes and inhibitors can be assessed to determine inhibitor potency and selectivity against native protein kinases, as well as hundreds of other ATPases. The ability to broadly profile kinase activities in native proteomes offers an exciting prospect for both target discovery and inhibitor selectivity profiling.